Notch signaling regulates a wide variety of mammalian cell fates and processes. The Notch signaling pathway is activated through direct cell-cell interactions that facilitate binding between the transmembrane ligand Delta on a signaling cell and the Notch receptor on a responding cell. Activation of this signaling pathway relies on Delta to induce Notch proteolysis for release of the biologically active Notch intracellular domain (NICD) that regulates expression of Notch target genes. Although the generation and activity of NICD has been intensively studied, little is known regarding the role and fate of the Notch extracellular domain (NECD) generated during Delta-induced Notch signaling. Studies have indicated that NECD removal is necessary for Notch proteolysis to occur; however, the mechanism of NECD release is not well defined. It has been proposed that NECD is released by ADAM shedding, but direct demonstration of ligand-induced NECD shedding has not been reported. Alternatively, NECD has been proposed to be removed through internalization into Delta cells, through a poorly characterized process of trans-endocytosis. However, evidence for transfer of NECD into Delta cells has so far only been obtained for Drosophila cells. The major aim of this proposal is to develop a system that will allow us to image and characterize transfer of NECD to interacting Delta cells using a mammalian cell co-culture assay. For these studies, we will use a combination of fluorophore-conjugated antibodies and tagged forms of Delta and Notch to localize transfer of Notch to co-cultured Delta cells using confocal microscopy. Given that the Notch extracellular domain accumulates in brains of CADASIL patients, understanding the fate of NECD is relevant to this inherited vascular dementia. Any information gained regarding the fate and role of the NECD during Notch signaling is expected to have biological and health related significance for understanding the molecular basis of stroke and dementia in CADASIL. Consistent with the NINDS Exploratory/Developmental Grant (R21) Program, experiments are proposed to initiate studies on the role of endocytosis in Delta induced Notch signaling to provide requisite data for longer-term, program specific funding (R01).